In the past decade, RNA interference (RNAi) has been described and characterized in organisms as diverse as plants, fungi, nematodes, hydra, and humans. Zamore and Haley (2005) Science 309, 1519-24. RNA interference in plants is commonly referred to as post-transcriptional gene silencing or RNA silencing and is referred to as quelling in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily conserved cellular defense mechanism used to prevent the expression of foreign genes and is commonly shared by diverse flora and phyla. Fire (1999) Trends Genet. 15, 358-363.
RNA interference occurs when an organism recognizes double-stranded RNA molecules and hydrolyzes them. The resulting hydrolysis products are small RNA fragments of 19-24 nucleotides in length, called small interfering RNAs (siRNAs) or microRNAs (miRNAs). The siRNAs then diffuse or are carried throughout the organism, including across cellular membranes, where they hybridize to mRNAs (or other RNAs) and cause hydrolysis of the RNA. Interfering RNAs are recognized by the RNA interference silencing complex (RISC) into which an effector strand (or “guide strand”) of the RNA is loaded. This guide strand acts as a template for the recognition and destruction of the duplex sequences. This process is repeated each time the siRNA hybridizes to its complementary-RNA target, effectively preventing those mRNAs from being translated, and thus “silencing” the expression of specific genes. Most plant miRNAs show extensive base pairing to, and guide cleavage of their target mRNAs. Jones-Rhoades et al. (2006) Annu. Rev. Plant Biol. 57, 19-53; Llave et al. (2002) Proc. Natl. Acad. Sci. USA 97, 13401-13406. In other instances, interfering RNAs may bind to target RNA molecules having imperfect complementarity, causing translational repression without mRNA degradation. The majority of the animal miRNAs studied so far appear to function in this manner.
Based upon the role of miRNAs as endogenous regulators of gene expression, substantial efforts have been made toward the design of miRNAs for targeted regulation of gene expression. For example, pre-miRNAs can be designed by replacing both the 21-nucleotide mature miRNA sequence and the complementary sequence (i.e., the miRNA* strand or miRNA star strand), with engineered or synthetic 21-nucleotide sequences. Such artificial pre-miRNAs have sequences identical to those of the natural pre-miRNAs except in the region encoding the mature miRNA and the star strand. By this method, artificial miRNAs (amiRNA) have been designed that can target and silence specific mRNA transcripts with complementary sequences.
Within miRNA sequences, highly conserved regions of 6-7 nucleotides, which are called seed sequences, are responsible for base pairing with a target gene/RNA. The seed sequences are positioned at nucleotides 2-7 or 2-8 by linear counting from the 5′-end of the miRNA molecule, while the remaining nucleotides are called non-seed sequences. miRNAs that are members of a same miRNA family (i.e., miRNAs with the same sequence at nucleotides 2-8) share the same predicted mRNA targets. See Bartel (2009) Cell 136, 215-233.
Given their role in sequence-specific gene regulation, siRNAs are envisioned to have many applications, including studies of gene function, development of therapies for conditions associated with aberrant protein expression or accumulation, and methods for conferring desirable traits, including in plants. To meet this need, the invention provides methods for efficient design of target-specific siRNAs, siRNA libraries, and siRNA molecules produced thereby, and methods for using the same.